High blood pressure is often associated with excessive salt and fluid retention from the kidney. The hormone angiotensin II (Ang II) is one of the most important factors in maintaining body salt and fluid and blood pressure homeostasis by regulating salt and fluid reabsorption from proximal tubules (PT) of the kidney. Thus increased production and actions of Ang II in PTs can cause salt and fluid retention and consequently increase blood pressure. Ang II exerts powerful effects on PT sodium and fluid transport by activating cell surface receptors on apical and basolateral membranes of PT cells. However, we have evidence that a) circulating and paracrine Ang II is taken up by PT cells in culture or by the kidney via an AT1 (AT1a) receptor-mediated mechanism; b) microinjection of Ang II directly into the cells can induce intracellular calcium responses; and c) intracellular Ang II can induce transcription of the major sodium and hydrogen antiporter, NHE-3, in isolated rat renal cortical nuclei. In this project, we hypothesize that apical (AP), rather than basolateral (BL), membrane AT1 (AT1a) receptors mediate the majority of intracellular uptake of Ang II by PT cells in vitro and in vivo, which involves the microtubule-dependent endocytic pathway. In vitro expression or intrarenal adenoviral transfer of an intracellular Ang II protein selectively in PT cells stimulates intracellular AT1 (AT1a) receptors to increase the expression and activity of NHE-3, promotes PT sodium and fluid reabsorption, and therefore induce hypertension. This hypothesis will be tested in four specific aims. Is Specific Aim I, we will test the hypothesis that in polarized PT cells, AP membrane AT1a receptors play a greater role in mediating intracellular uptake of extracellular Ang II than BL membrane AT1a receptors in vitro and in vivo. In the absence of apical AT1a receptors, AT1b receptors or the endocytic receptor megalin may partially assume the role of AT1a receptors. In Specific Aim II, we will test the hypothesis that in polarized PT cells, AP membrane AT1a receptor-mediated intracellular uptake of Ang II is mediated by two major endocytic motifs of AT1a receptors in the cytoplasmic tail, and is regulated by the microtubules- and lipid rafts/caveolin-1 (CAV-1)-dependent mechanisms.. In Specific Aim III, we will test the hypothesis that in polarized PT cells, in vitro expression of an intracellular Ang II protein, ECFP/AII, increases the expression and the activity of NHE-3 in AP membranes via the activation of [Ca2+]i/PKC1/2II, MAP kinases ERK1/2, and NF-:B signaling pathways. Finally, Specific Aim IV will test the hypothesis that intrarenal adenoviral gene transfer of an intracellular Ang II protein selectively in PTs increases salt and fluid reabsorption, promotes salt and fluid retention, and thereby induce hypertension by increasing the expression and activity of apical NHE-3 via activation of AT1 (AT1a) receptors. These studies will provide novel insights into the important roles and underlying cellular and molecular mechanisms of intracellular Ang II in the physiological regulation of salt and fluid reabsorption in proximal tubules of the kidney and in the pathogenesis of hypertension. PUBLIC HEALTH RELEVANCE: About one in three U.S. adults will develop hypertension or hypertension-related complications in their life time, and treating hypertensive diseases costs the U.S. economy several hundreds of billion dollars a year. Salt and fluid retention due to the actions of the hormone angiotensin II remains one of the most important factors in the development and progression of hypertension. This project investigates the signaling mechanisms that regulate the uptake of angiotensin II by proximal tubule cells of the kidney and studies how internalized angiotensin II acts to increase salt and fluid reabsorption from proximal tubules, promote body salt and fluid retention, and therefore cause hypertension. The new information generated by this project will help us better understand renal mechanisms of hypertension and develop new drugs to treat hypertension.